弥漫大B细胞淋巴瘤信号通路相关耐药microRNA和蛋白分子的筛选及功能研究

Diffuse large B cell lymphoma is a highly heterogeneous tumor, although R - CHOP scheme has improved the total effective rate of the tumor, there are still a third of the patients with primary resistance or eases died of tumor recurrence. Primary or acquired drug resistance is a key factor in limiting effect. Multi-channel and more targets signaling pathways are abnormal, signaling network regulation is out of control, apoptosis, cell proliferation, these are important drug resistance mechanism of DLBCL. How to resistant reversal experiments for molecular targets of drug target in the signal pathway is still difficult. Research shows that: the NF-kappa B, PI3K/AKT, PKC, and Wnt/beta - catenin signaling pathway mediate resistance, we want to screen signaling pathways-associated resistance miRNA using miRNA expression profile chip , and carry out an differential proteomics analysis on the chemosensitivity of DLBCL tissue with high-throughput screening of resistance-associated protein，we will analysis bioinformatics, verify histology and function research for screening the chemotherapy drug resistance related miRNAs and protein from the mRNA and protein level , we are expected to find the miRNA and protein associated with the resistance to chemotherapy and provide new ideas for screening molecular markers of tumor resistance and reversal resistant to targeted therapy．

弥漫大B细胞淋巴瘤是高度异质性肿瘤，尽管R-CHOP方案提高了该肿瘤的总有效率，仍有1/3患者出现原发耐药或缓解后复发而死于肿瘤。原发和获得性耐药是限制疗效的关键因素。多通道多靶点信号传导通路异常、信号网络调控失衡、细胞增殖失控、凋亡受阻，是DLBCL耐药的重要机制。如何以信号通路中的分子靶点作为药物靶标进行耐药逆转实验研究仍有困难。研究表明：NF-KB、PI3K/AKT、PKC、Wnt-/β-catenin等信号通路介导耐药，本组拟选择不同耐药细胞株，用miRNA表达谱芯片筛选信号通路相关耐药microRNA，RT-PCR验证耐药相关miRNA对肿瘤细胞耐药性影响；用蛋白质组学技术分离鉴定化疗敏感性不同DLBCL组织中差异表达的与信号通路相关的蛋白质，对其进行生物信息学分析，进一步进行组织学验证及功能研究，为从mRNA及蛋白水平筛选肿瘤耐药分子标志物及逆转耐药的靶向治疗奠定基础。

弥漫大B细胞淋巴瘤（DLBCL)是成人最常见的B细胞淋巴瘤,经标准化治疗后仍有30~40%的患者治疗欠佳短期内复发耐药甚至死亡。研究发现微小RNA（miRNA）在DLBCL中发挥重要作用；蛋白质作为机体最终功能的执行者其异常表达可引起肿瘤，筛选及验证异常表达蛋白质成为研究热点；亦有研究指出多重信号通路所涉及DLBCL发病机制和寻找信号通路相关潜在治疗靶点成为提高DLBCL患者疗效的关键。我们选择人新鲜冰冻DLBCL组织为实验组，人淋巴结反应性增生为对照组，应用miRNA表达谱芯片和蛋白质组学筛选差异miRNA和蛋白质，对差异miRNA和蛋白质进行生物信息学分析，最后对已筛选PI3K/AKT信号通路中的差异miRNA、mRNA和蛋白质进行验证及功能研究。我们筛选出204个差异miRNA，54个上调，150个下调；差异miRNA预测7522个靶基因；靶基因富集在Pathway in cancers、MAPK、Wnt、PI3K/AKT等信号通路；qRT-PCR验证PI3K/AKT信号通路中的9个差异miRNA表达，发现hsa-miR-193a-3p、hsa-miR-19b-3p、hsa-miR-370-3p、hsa-miR-28-5p，hsa-miR-490-5p在DLBCL中低表达，hsa-miR-630在DLBCL高表达。筛选出335个差异蛋白质，131个上调，204个下调；差异蛋白质富集在Pathway in cancers、PI3K/AKT、Alcoholism、Necroptosis等信号通路；PRM初步验证PI3K/AKT 信号通路中的9个差异蛋白质的蛋白表达水平，发现DLBCL中TCL1、HSP90、14-3-3ζ、IKKβ表达上调，Gβγ2、LAMα5、LAMβ2、CO1α1表达下调；免疫组化染色发现DLBCL组织中TCL1阳性表达率为68.6%；相关性分析发现TCL1表达与AKT相关；生存分析发现TCL1过表达是DLBCL患者短的总生存期和无进展生存期的独立影响因素。此次研究在miRNA、mRNA、蛋白质水平筛选和验证与DLBCL发生发展相关的可能致病因子和信号通路，对已筛选和验证调控重要信号通路的致病因子进行功能研究，为深入理解DLBCL的发生发展、提供新新的治疗靶标奠定理论基础。